Orientador: Jose Roberto Bosqueiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T10:10:15Z (GMT). No. of bitstreams: 1 Roma_LeticiaPrates_M.pdf: 861159 bytes, checksum: 232359e1de6e654ce6bb13758ca58bd2 (MD5) Previous issue date: 2006 / Resumo: As células B pancreáticas possuem controle multifatorial que permite a secreção de insulina em quantidade e tempo adequados. Os glicocorticóides modulam a secreção de insulina dependendo do tempo e dose em que forem utilzados. Assim,o presente trabalho teve por objetivo analisar as alterações na expressão gênica e protéica de ilhotas de ratos tratados com dexametasona (1mg/kg, 5dias). Utilizando a técnica de cDNA Macroarray observamos que dos 1176 genes presentes na membrana, 66 tiveram sua expressão aumentada e 38 genes tiveram sua expressão diminuída. Os genes com expressão aumentada pertencem às vias de estresse celular (JNK1), inibidores do ciclo celular (p21) e vias de apoptose (Baxa e Fas). Os genes com expressão diminuída pertencem ao ciclo celular (ciclinas D1 e D2, CDK4) e vias de sinalização PI3K, AKT1 e P70. Demonstramos também aumento na expressão protéica da Bax a, redução na expressão da proteína anti-apoptótica Bcl-2, PI3K e P70. Animais tratados com dexametasona por 5 dias possuem níveis plasmáticos aumentados de insulina, triglicérides e ácidos graxos livres. Ilhotas isoladas de animais tratados com dexametasona por 5 e 10 dias apresentaram maior secreção de insulina em relação aos controles, em concentrações basais e estimulatórias de glicose e 40mM de potássio. Porém, o tratamento por 10 dias com dexametasona induziu diminuição na secreção de insulina quando comparado aos animais tratados por 5 dias. Nossos dados sugerem o tratamento com dexametasona pode modular (direta ou indiretamente) a expressão de diversos genes e proteínas envolvidas na apoptose e sobrevivência de células na ilhota pancreática. Essa modulação pode, em longo prazo, se refletir na secreção de insulina como visto nos animais tratados por 10 dias / Abstract: Insulin secretion from pancreatic B-cells is regulated by nutrients like glucose and amino acids and by neurotransmitters, and hormones. Since glucocorticoids modulate insulin secretion we investigated the effects of dexamethasone on gene and protein expression in pancreatic islets from rats treated with the glucocorticoid for 5 days (mg/kg/day). Using cDNA array analysis we found that, out of 1176 genes presented in the array, 66 were up-regulated and 38 down-regulated after dexamethasone treatment. RT-PCR confirmed the macroarray results for 4 genes whereas the expression of these transcripts was confirmed by Western blotting for the corresponding proteins. Many of the up-regulated genes are implicated in apoptosis (Bax a, Fas), cell cycle regulation (p21) and stress response (JNK1) whereas many of the down-regulated were involved in cell cycle progression (cyclins D1 and D2, and CDK4), and survival and proliferations pathways (PI3K, AKT, P70). The protein expression of Bax a was increased whereas Bcl-2, PI3K and P70 repressed. The rats treated with dexamethasone for 5 days showed higher insulin, triglicerides and free fatty acids plasma levels than controls. The insulin secretion, in response to glucose and high concentrations of K+, in islets isolated from dexamethasone-treated rats for 5 and 10 days was higher than control rats. However, after 10 days of treatment with dexamethasone the insulin secretion was lower than after 5 days, but still higher than controls. In conclusion, these data indicate that dexamethasone-treatment (directly or indirectly) modulates the expression of several genes and proteins involved in apoptosis and survival of pancreatic islet-cells, and could, thereafter, modulates the insulin secretion in rats treated for 10 days / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Orientador: Everardo Magalhães Carneiro / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T09:09:25Z (GMT). No. of bitstreams: 1 Ribeiro_RosaneAparecida_D.pdf: 2339564 bytes, checksum: 146ea5a2123d76628db5f4758417e020 (MD5) Previous issue date: 2009 / Resumo: Neste estudo, investigamos os efeitos da suplementação com taurina (TAU; 2% adicionada à água de beber) sobre a tolerância à glicose e a secreção de insulina frente a diferentes secretagogos em camundongos adultos. Camundongos suplementados apresentaram aumento da tolerância à glicose e da sensibilidade à insulina. Ilhotas isoladas destes animais secretaram mais insulina em resposta à glicose e L-leucina. A oxidação da L-leucina foi maior no grupo TAU, não havendo diferenças quanto ao consumo de glicose, concentrações de ATP e expressão do transportador da glicose (GLUT) 2 e da glicoquinase (GCK). A captação de Ca2+, na presença de glicose, e a expressão protéica da subunidade ß2 do canal de Ca2+ sensível à voltagem foi maior no grupo TAU comparado ao controle (CTL). Ainda, a expressão protéica da PL (fosfolipase) C ß 2 e da PK (proteína quinase) Aa, bem como a secreção de insulina em resposta a agentes potencializadores tais como carbacol (Cch) e IBMX, foi maior nas ilhotas TAU. A mobilização intracelular de Ca2+ induzida por Cch foi também maior em ilhotas deste grupo, e observamos que a inibição da PKA reduziu a captação de Ca2+ em resposta à glicose no grupo suplementado. Além disso, ilhotas TAU secretaram mais glucagon em relação a ilhotas CTL, quando em presença de baixa concentração de glicose. Concluindo, a suplementação com TAU melhora a homeostase glicêmica e aumenta a secreção de insulina de ilhotas isoladas e incubadas na presença de nutrientes e agentes potencializadores da secreção. Os efeitos sobre a secreção estão relacionados ao melhor manejo dos íons Ca2+ pelas células insulares provenientes dos animais suplementados com TAU. / Abstract: In this study, we investigated the effects of taurine (TAU)-supplementation (2% in the drinking water) on glucose tolerance and insulin secretion stimulated by different secretagogues in adult mice. TAU-supplemented mice showed enhanced glucose tolerance and insulin sensitivity when compared to controls (CTL). In addition, their islets secreted more insulin in response to high concentrations of glucose and L-leucine. L-[U-14C]leucine oxidation was higher in TAU islets compared with CTL islets, whereas D-[U-14C]glucose oxidation, ATP levels, and the protein expression of the glucose transporter (GLUT) 2 and of glucokinase (GCK) were similar. 45Ca uptake induced by high glucose concentrations was increased in TAU islets as well as the expression of the ß2 subunit of the L-type Ca2+ channel. In addition, the insulin secretion induced by carbachol (Cch) and IBMX, but not, by forskolin and PMA was higher in TAU-supplemented compared with CTL islets. The higher insulin secretion in the presence of Cch is accompanied by an increase in the expression of PL (phospholipase) C ß 2 protein and a higher intracellular Ca2+ mobilization. Besides, TAU-supplemented islets showed increased PK (protein kinase) Aa expression. Since the increase in Ca2+ uptake induced by glucose in TAU islets was minimized by the presence of the PKA inhibitor, H89, this kinase seems to be important for the better Ca2+ handling in these islets. TAUsupplementation also turns the a-cells more sensitivity since these cells secreted more glucagons compared with CTL islets. In conclusion, TAU supplementation enhances glucose tolerance and insulin sensitivity in mice and turns the islets more sensitive to nutrients and to potentiators of secretion. The effect on insulin secretion seems to be linked to a better Ca2+ handling by ß-cells. / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Orientador: Antonio Carlos Boschiero / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T03:49:57Z (GMT). No. of bitstreams: 1 Silva_KellyElizeuda_M.pdf: 1866524 bytes, checksum: 87ae185f2da61d551d322f4375943e54 (MD5) Previous issue date: 2007 / Resumo: O INGAP-PP é um pentadecapeptídeo constituído pela seqüência de aminoácidos do resíduo 104 ao 118 do INGAP (Islet Neogenesis Associated Protein), o qual é expresso no pâncreas exócrino e também nas células ductais durante a neogênese de células ßpancreáticas. Nesse trabalho, analisamos o efeito do tratamento crônico (4 dias) com INGAP-PP sobre a secreção de insulina, expressão gênica e protéica das subunidades Kir6.2 e SUR1 que formam o canal de K+ATP e do fator de transcrição Foxa2 (regulador da expressão das subunidades desse canal), em ilhotas isoladas de ratos adultos. As ilhotas tratadas com INGAP-PP (10 µg/mL) secretaram significativamente mais insulina quando incubadas por 1 h em concentrações entre 2.8 e 22.2 mM de glicose em comparação às ilhotas controles. Resultados de RT-PCR mostram que ilhotas tratadas com INGAP-PP tiveram expressão gênica do Foxa2 e das subunidades SUR1 e Kir6.2 aumentada. A expressão das proteínas SUR1 e Foxa2, analisada por Western Blotting, também foi maior nas ilhotas tratadas com INGAP-PP. Quando perfundidas na presença de 22,2 mM de glicose o aumento da secreção de insulina pelas ilhotas tratadas se manifestou com um primeiro pico secretor significativamente maior do que as ilhotas controles. Em presença de 2,8 mM de glicose, ilhotas tratadas com INGAP-PP secretaram mais insulina frente à concentrações despolarizantes de KCl ou tolbutamida (100 µM). Entretanto, a secreção de insulina estimulada por tolbutamida não diferiu entre os grupos em presença de 22,2 mM de glicose. A análise do efluxo de 86Rb mostrou que as ilhotas cultivadas com INGAP-PP apresentam menor efluxo do isótopo em relação às controle. Portanto, a maior secreção de insulina frente à glicose e concentrações despolarizantes de K+ indica que o tratamento com INGAP-PP induziu alterações que tornaram as células ßmais sensíveis a agentes despolarizantes. Quando associamos estes resultados ao aumento da expressão das proteínas formadoras do canal K+ATP e à redução do efluxo de 86Rb pelas ilhotas tratadas com INGAP-PP, podemos sugerir que o aumento no número de canais KATP pode ser um dos responsáveis pelo aumento na secreção de insulina nas ilhotas tratadas com o peptídeo / Abstract: Cultured adult rat islets were used to study the effect of INGAP-PP upon: a) gene expression of Kir6.2 and SUR1 of K+ATP channels and of their transcription factor Foxa2 (RT-PCR), b) protein levels (Western blotting) of SUR1 and Foxa2, c) static and dynamic insulin secretion elicited by metabolic and non metabolic stimuli and d) 86Rb efflux from perifused islets. INGAP-PP increased significantly the expression of Kir6.2, SUR1 and Foxa2 and the protein levels of SUR1 and Foxa2. Islets cultured with INGAPPP and further incubated for 1 h with 2.8 mM glucose, significantly enhanced the release of insulin in response to 40 mM KCl, and 100 µM tolbutamide. The dose-response curve of insulin secretion to increasing glucose concentrations (2.8 to 22.2 mM) shifted to the left in INGAP-PP-cultured islets with an EC50 of 10.0 ± 0.4 vs. 13.7 ± 1.5 mM glucose of the controls (P < 0,05). In dynamic studies INGAP-PP increased significantly the first-phase of insulin secretion elicited by either 22.2 mM glucose or 100 µM tolbutamide and promotes a higher glucose-induced reduction of 86Rb efflux from perifused islets. These results confirm the enhancing effect of INGAP-PP upon insulin release induced by different secretagogues and provide new evidence that such effect is due, at least partly, to an enhanced expression of the SUR1 and Kir6.2 genes of K+ATP channels and of the Foxa2 gene that controls their expression. They would also suggest that INGAP-PP could potentially be used to maintain the capacity of cultured islets to release insulin in response to glucose and maybe for the treatment of diabetes / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

In the studies about ovarian cancer cells, basal iNOS expression in the chemosensitive OV2008 cells was significantly higher than in the chemoresistant C13* cells. Cisplatin further increased iNOS expression in OV2008 cells, but had no effect in C13* cells. Furthermore, cisplatin dramatically reduced the expression levels of eNOS and nNOS, but again only in OV2008 cells. The data suggest that failure of cisplatin to upregulate iNOS and downregulate eNOS and nNOS in C13* cells could be an etiological factor in chemoresistance. Addition of exogenous NO at high levels, using SNAP, significantly increased p53 protein levels and caused apoptosis in both cell types. Specific iNOS inhibitor (1400W) partially blocked the pro-apoptotic effects of cisplatin in OV2008 cells, suggesting involvement of iNOS in cisplatin-induced apoptosis. However, blocking of all three isoforms of NOS with NG-amino-L-arginine in C13* cells dramatically changed these cells from chemoresistant to chemosensitive, greatly potentiating the pro-apoptotic effects of cisplatin. / Inhibition of Src-kinase activity reduces DNA synthesis in ovarian cancer cells. In an in vitro experiment, Src phosphorylated PKG on a tyrosine residue and PKG, presumable via serine-phosphorylation of Src, enhanced Src auto(tyrosine)phosphorylation. In ovarian cancer cells, inhibition of basal PKG activity with DT-2 decreased both basal and EGF-stimulated Src kinase activation and DNA synthesis. The data suggest that PKG at basal activity, is necessary for both basal and growth factor-stimulated Src kinase activation and enhanced DNA synthesis in human ovarian cancer cells. / The novel role of sGC/cGMP/PKG pathway on stimulating cell proliferation, potentially via interaction with the Src kinase pathway in human ovarian cancer cells, was demonstrated. ODQ dramatically reduced DNA synthesis rates, suggesting that basal sGC activity and basal cGMP levels are needed for ovarian cancer cell proliferation. DT-2 also reduced cell proliferation, suggesting the direct involvement of PKG. ANP and BNP had no effect on cell proliferation, suggesting that further activation of cGMP/PKG pathway above basal levels does not further enhance cell proliferation. / The present study also demonstrated that elevating cGMP slightly above the basal levels further protects pancreatic islet cells against spontaneous onset of apoptosis. The results showed that natriuretic peptides (both ANP and BNP) and low-level NO (i.e. physiological levels) as supply by NO donor, S-nitroso-N-acetylpenicilamine (SNAP) further prevented spontaneous apoptosis in pancreatic islets after isolation, whereas NO at high concentrations (i.e. pathological levels) promoted apoptosis in pancreatic islet cells. The commonly-used PKG inhibitor KT5823 and the newly-developed specific PKG inhibitor DT-2 completely prevented anti-apoptosic effect of ANP, suggesting the direct involvement of PKG in protection against spontaneous apoptosis. / The present study demonstrated that basal activity of sGC/cGMP/PKG signaling pathway is essential for partially limiting spontaneous apoptosis in pancreatic islet cells. The sGC inhibitor ODQ caused induction of apoptosis, which was completely blocked by co-treatment with ANP or BNP, agents that elevate cGMP via pGC, bypassing the ODQ block. Co-treatment with 8-Br-cGMP, a direct activator of PKG also completely prevented ODQ-induced apoptosis in islets. / Leung Lai-han. / "July 2006." / Adviser: Ronald Ray Fiscus. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1483. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 175-191). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

在2型糖尿病病人身上，常常併發高膽固醇血症，HMG CoA 還原酶的抑制劑常常用作治療這類病症。由於高膽固醇血症與胰島素抵抗和2型糖尿病有著密切關係，我們推測辛伐他汀對於2型糖尿病的發展有著保護和有利的作用。在這項研究中，我們主要測試了辛伐他汀 （10 nM； 24 hr）對於胰島β細胞主要功能的影響，包括其對於葡萄糖的胰島素分泌功能影響。我們假設，在肥胖/糖尿病（db⁺/ db⁺）小鼠分離的胰島，辛伐他汀可以恢復葡萄糖 （5 mM和15 mM）引起的胰島素分泌（加上降低的胰島素含量）。 / 在這個項目中，我們運用24周大的基因糖尿糖C57BL/KSJ +db/+db (db⁺/db⁺)肥鼠和相同年齡的無糖尿病C57BL/KSJ +m/+m (db⁺/m⁺)小鼠作為動物模型。通過應用obese/diabetic (db+/db+)和lean/non-diabetic (db+/m+)中分離的胰腺胰島和胰島β細胞，我們研究了胰腺胰島功能性障礙的潛在機理以及辛伐他汀對於恢復葡萄糖 （5 mM和15 mM）引起的胰島素分泌（加上降低的胰島素含量）的有利作用。資料清晰的顯示，葡萄糖引起的胰島素分泌和胰島素含量在obese/diabetic (db+/db+)的胰腺胰島中明顯低於在lean/non-diabetic (db⁺/m⁺)的胰腺胰島中。在24hr的辛伐他汀處理後，辛伐他汀恢復了葡萄糖 （5 mM和15 mM）引起的胰島素分泌（加上降低的胰島素含量）及葡萄糖 （15 mM）引起的胞內鈣離子變化。 / 在這個項目中，我們證明鈣敏感受體 （CaSR）在obese/diabetic (db⁺/db⁺)中的表達量明顯較低，而辛伐他汀的處理可以顯著性增加鈣敏感受體在obese/diabetic (db⁺/db⁺)胰島中的表達。有人建議說，obese/diabetic (db⁺/db⁺)的胰島中被抑制的鈣敏感受體表達與胰島β細胞的胰島分泌功能障礙有關。這暗示了辛伐他汀可能通過變構啟動鈣敏感受體來恢復obese/diabetic (db⁺/db⁺)胰島中葡萄糖引起的胰島素分泌和胰島含量。實驗也同樣証明辛伐他汀調節的PLA₂信號通路對於辛伐他汀改善obese/diabetic (db⁺/db⁺)胰島β細胞的胰島素分泌功能起著至關重要的作用。除此之外，我們的實驗結果證明高濃度的葡萄糖處理顯著的增加了obese/diabetic (db⁺/db⁺)細胞膜肌動蛋白骨架的密度，而辛伐他汀顯著的減少了這一變化。因此，obese/diabetic (db⁺/db⁺)胰島β細胞的胰島素分泌障礙是由肌動蛋白細胞骨架聚集阻礙胰島素顆粒胞吐引起的。而辛伐他汀通過解聚和重組肌動蛋白細胞骨架來改善obese/diabetic (db⁺/db⁺)胰島β細胞的胰島素分泌功能。 / 在這項研究中，我們的實驗結果證明葡萄糖可以顯著提高obese/diabetic (db⁺/db⁺)胰島β細胞內ROS的含量。而辛伐他汀處理部分降低了胰島β細胞內ROS的含量。除此之外，我們還研究了5 mM和15 mM葡萄糖對於內質網應力（ER-stress）相關的蛋白比如PERK, eIF2α 和IRE1表達的影響。這些內質網跨膜蛋白可以感應ER-stress從而啟動應力感測器來開啟複雜的信號通路。與lean/non-diabetic (db⁺/m⁺)相比，PERK and eIF2α在obese/diabetic (db⁺/db⁺)的胰島中表達量更低，這表明obese/diabetic (db⁺/db⁺)胰島β細胞的功能性障礙可能與ER-stress有關。而辛伐他汀的處理明顯的增加了這些蛋白的表達量，由此證明辛伐他汀還通過對抗ER-stress來保護obese/diabetic (db⁺/db⁺)胰島β細胞。 / 總而言之，我們的資料第一次證明了辛伐他汀通過PLA₂信號通路變構啟動鈣敏感受體來保護obese/diabetic (db⁺/db⁺)胰島β細胞（比如：恢復葡萄糖引發的胰島素分泌和提高減少的胰島素含量），還通過提高obese/diabetic (db⁺/db⁺)胰島β細胞中被抑制的ER-stress相關蛋白的表達量來抵抗ER-stress帶來的損傷。 / Diabetics often have hyperlipidemia as a co-morbidity. Despite the well-documented cholesterol-lowering properties of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) in treating hypercholesterolemia, the beneficial effects of statins consumption in T2DM treatment are confusing. In the current study, we examined the effects of the simvastatin (10 nM; 24 hr) on β-cell function leading to insulin secretory response to glucose. We hypothesized that statins restore the blunted glucose (5 mM and 15 mM)-induced insulin secretion (plus the reduced insulin content) of isolated pancreatic islets of obese/diabetic (db⁺/db⁺) mice. / In the present study, genetically diabetic C57BL/KSJ +db/+db (db⁺/db⁺) mice at 24 week of age and their age-matched non-diabetic littermates C57BL/KSJ +m/+m (db⁺/m⁺) were used. Our results clearly showed that the suppressed glucose (5 mM and 15 mM)-induced insulin release (plus insulin content) and glucose (15 mM)-induced [Ca²⁺]i changes of isolated pancreatic islets of obese/diabetic (db⁺/db⁺) was restored after simvastatin (10 nM; 24 hr) treatment. / The biochemical existence of CaR in pancreatic islets of lean/non-diabetic (db⁺/m⁺) and obese/diabetic (db⁺/db⁺) mice was confirmed. The suppressed/down-regulated expression of CaR was associated to the blunted insulin secretion in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice, and it was markedly up-regulated by simvastatin (10 nM; 24 hr). The involvement of CaR-mediated PLA₂ signaling in simvastatin (10 nM; 24 hr)-induced restoration of glucose (15 mM)-induced insulin secretion in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice was investigated. Our results also showed that the increased density of plasma membrane actin cytoskeleton of obese/diabetic (db⁺/db⁺) mice was significantly decreased by simvastatin (10 nM; 24 hr) treatment. The simvastatin-induced depolymerization and remodeling of actin cytoskeleton may improve insulin secretion capability in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice. / The glucose (15 mM)-induced intracellular ROS level was significantly higher in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice. The elevated ROS level was partially diminished by simvastatin (10 nM; 24 hr) treatment. The protein expressions of PERK and eIF2α (ER stress proteins) were lower in pancreatic islet cells isolated from obese/diabetic (db⁺/db⁺) mice, suggesting that abnormal expresstion/activity of PERK and eIF2α would be coupled to the ER-stress mediated failure of pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice. As simvastatin (10 nM; 24 hr) up-regulated the protein expression of these proteins, this drug exerted protective effect on pancreatic β-cells against ER stress and restored the blunted glucose (15 mM)-induced insulin secretion (plus the reduced insulin content) in obese/diabetic (db⁺/db⁺) mice. / In conclusion, our results demonstrate, for the first time, that simvasatatin (a HMG-CoA reductase inhibitor) (10 nM; 24 hr) provides beneficial effects (i.e. restoration of the blunted glucose-induced insulin release plus the reduced insulin content) in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice via the allosteric modification/up-regulation of extracellular calcium-sensing receptor through the PLA₂ signaling pathway, and provides protective/antioxidant effects against oxidative stress caused by chronic hyperglycemia in pancreatic β-cells of obese/diabetic (db⁺/db⁺) mice by up-regulating protein expression of the suppressed ER stress sensors and antioxidant enzyme. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Au, Lai Shan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 458-532). / Abstracts also in Chinese.

胰腺內的胰島具有極其重要的功能，通過產生并分泌一系列的胰島荷爾蒙，特別是能控制機體葡萄糖利用的胰島素，來調節體內血糖穩態。胰島素的分泌受到多種因素或信號通路的調節。据信，在臨床上表現出來明顯的高血糖症的時候，胰島細胞的分泌功能已經出現典型性的缺陷。由此，大量的研究證據指出，2 型糖尿病表現出來的代謝型缺陷主要為胰島功能紊亂，而并不是周圍組織胰島素抵抗。這表明，胰島素功能缺陷是早於高血糖症的發生的。另一方面，大量證據表明長期性的高血糖會導致胰島　細胞功能紊亂。鑒於此，揭示胰島功能調節的潛在機理并闡明胰島功能与高血糖症之間的關係變得尤為重要。 / 在臨床上表現出能調節胰島功能和血糖控制的相關因子正與日俱增。其中極具研究價值的是一種多肽，稱作胰高血糖素様肽（GLP-1），其作用表現在通過增強胰島素分泌和胰島素敏感性來增強胰島　細胞的功能和增值。GLP-1 在體內的降解能被DPP-4 的抑製劑所延阻。同時，通過對一種名為SGLT2 的葡萄糖轉運蛋白的抑制，機體內的血糖水平能被顯著降低。這一作用是通過阻止腎臟對葡萄糖的重吸收來實現的，並且是不依賴于胰島素的。由於DPP-4 抑制所表現的最終生理作用需要通過胰島素的信號通路來實現，但SGLT2 的抑制卻不依賴於胰島素，由此不難想象，對SGLT2 和DPP-4 的聯合抑制在2 型糖尿病的血糖控制方面具有潛在的協同效應。即通過對SGLT2 的抑制來顯著降低血糖水平，從而促進GLP-1 在體內的作用效應。因此，本研究的第一部分研究SGLT2 和DPP-4 的單一或聯合抑制（利用SGLT2 抑製劑BI-38335 和DPP-4 抑製劑linagliptin）在二型糖尿病動物模型db/db老鼠種對胰島功能和體內葡萄糖穩態的作用。在此研究中，我們比較了SGLT2 和DPP-4 單一抑制或聯合抑制對db/db 老鼠胰島功能的影嚮。研究發現，所有的實驗組都能顯著降低血糖以及糖化血紅蛋白（HbA1c）的水平，而且聯合抑制組表現出更叫顯著的效應。聯合抑制組增強了胰島細胞的胰島素分泌功能，改善葡萄糖耐受并增加胰島素的敏感性。於此一致的是，聯合抑制組降低了β細胞凋亡和胰島免疫細胞標記物，並且抑制了与TLR2 信號通路相關的一系列炎症分子，通過則一系列作用實現對胰島的保護。上述研究表明，對SGLT2 和DPP-4 的聯合抑制在對胰島功能和胰島形態學上的保護至少能夠表現出加性效應，從而更好實現對血糖的調控。 / 在第一部分的工作中，我們利用的動物模型db/db 老鼠是一類較嚴重的糖尿病動物模型，它表現出及其嚴重的高血糖症，糖耐受失調同β細胞缺陷。我們集中于研究SGLT2 和DPP-4 的抑制對這類嚴重糖尿病的胰島功能的調節，具體表現在對胰島β細胞功能的正向調節，包括胰島素分泌功能的增強和β細胞質量的增加。廣為接受的一點是，胰島素抵抗和胰島素分泌功能的缺失最能表徵從正常葡萄糖耐受發展到2 型糖尿病的這一進程。這一進程的早期主要表現為由肥胖或衰老而引起的代償性的胰島素抵抗，此時伴有正常或受損的葡萄糖耐受以及正常的胰島素分泌功能。此時，任何能影響胰島功能的因素都會減緩或加速2 型糖尿病的發生。鑒於此，研究此类因素從而到达阻止2 型糖尿病的发生就显得尤为重要。因此，在本研究的第二部分，我们研究利用高脂飼料诱导的肥胖老鼠模型和老化的老鼠模型来分别研究煙酸（niacin 或 nicotinic acid）对胰岛功能的影響。煙酸是一種臨床上廣汎使用的降血脂藥物，但近年來的研究發現長期或高劑量的使用會導致高血糖症和血糖控制失調的出現，然而這一現象產生的具體機製並不清楚。因此，我們第二部分的研究集中於揭示煙酸引起的高血糖症是否歸因於其對胰島功能的破壞，以及潛在的分子機制。我們的研究發現，在肥胖老鼠和老齡鼠中，煙酸能夠引起高血糖症，破壞葡萄糖體內穩態並且降低胰島素分泌能力；另一方面，煙酸增加饑餓血清胰島素水平並且引起葡萄糖耐受實驗中第一期胰島素分泌缺陷。體內和體外實驗還發現煙酸誘導煙酸受體GPR109a，UCP2 和PPARγ的表達增加以及SIRT1 的表達和NAD,NAD/NADH 降低。通過基因沉默技術降低GPR109a 在β細胞中的表達，我們發現煙酸的上述作用都被極大的減弱，從而揭示了煙酸引起的胰島功能降低是由其受體GPR109a 介導的。 / 總闊來說，我們的研究揭示了DPP-4 同SGLT2 的聯合抑制在增強胰島功能和胰島形態學上的保護以及改善胰島素抵抗等方面能夠表現出加性效應，從而更好實現對血糖的調控。另一方面，我們的研究闡述了煙酸通過它的受體GPR109a 以及其下游信號通路如PPARγ和SIRT1 來損害胰島細胞功能。綜上所述，我們當前的研究證實了一系列因素對胰島功能的調控，從而充實并擴展了我們對胰島功能和血糖控制以及2 型糖尿病之間關係的認識。 / Pancreatic islets are of great importance to govern glucose homeostasis through production and secretion of islet peptide hormones, notably insulin, which functions as a master regulator to control glucose disposal in the body. Insulin secretion is regulated by various factors and signaling pathways. It is well known that islet insulin secretory function is typically lost by the time when signs of hyperglycemia that becomes clinically apparent. Thus, it has been pointed out that islet dysfunction, rather than peripheral insulin resistance, is the primary defect of type 2 diabetes mellitus (T2DM), indicating that deficiencies in islet function are prior to the onset of hyperglycemia. On the other hand, it is also widely accepted that chronic hyperglycemia results in islet β cells dysfunction. In this regard, it is of great importance to unravel the underlying mechanisms by which islet function is regulated, thus elucidating the relationship between hyperglycemia and islet function. / There are ever increasing candidates of clinically relevant factors identified as criticalregulators for islet function and glycemic control. Of great interest is the glucagon-like peptide 1 (GLP-1) that improves β cell function and proliferation and its degradation can be delayed by dipeptidyl peptidase-4 (DPP-4) inhibition. Meanwhile, plasma glucose levels can be remarkably lowered by inhibition of sodium-glucose co-transporter 2 (SGLT2), through blockade of renal glucose reabsorption. In this regard, since the mode of action of SGLT2 inhibition is independent of insulin but the efficacy of DPP-4 inhibition relies on the insulin signalling, it is plausible to hypothesize that sustained lowering of plasma glucose by SGLT2 inhibition can facilitate the actions of GLP-1 from DPP-4 inhibition, thus leading to a potential synergistic effect on islet function and glycemic control. Accordingly, the first part of this study was to investigate the combination effects of SGLT2 and DPP-4 blockade on islet function and glucose homeostasis using an animal model of T2DM, the db/db mice. We compared the effects of either DPP-4 inhibition (by a DPP-4 inhibitor, linagliptin) or SGLT2 inhibition (by an SGLT2 inhibitor, BI-38335) individually and in combination on islet function and glycemic control in db/db mice. Active treatments markedly enhanced islet function, improved glycemic control and reduced islet and peripheral tissue inflammation, with the combined treatment showing the greater effects. These data indicate that combined SGLT2 inhibition with DPP-4 inhibition work additively to exhibit benefits to islet function, inflammation and insulin resistance, thus improving glycemic control. / In the first part, we investigated a positive regulation of islet function in overt diabetic mice, in which there are severe hyperglycemia and β cell failure. It is widely accepted that the progression from normal glucose tolerance to T2DM is characterized by dual defects that include insulin resistance and an insulin secretory defect caused by β cell dysfunction. In the early stage, there is compensated insulin resistance resulting from obesity or aging with normal or even impaired glucose tolerance as well as nearly normal insulin secretory capacity. As such, any factors that affect islet function in this stage may delay or accelerate the onset of diabetes. In this regard, it is noteworthy to study the regulation of such factors in islet function in order to prevent the development of T2DM. Thus, in the second part, we investigated how islet function was regulated by a widely used lipid-lowering drug, niacin (nicotinic acid), in obese mice and aged mice. Niacin has been known to impair euglycemic control during prolonged and high dose treatments but the underlying mechanism(s) whereby the islets are involved remains unclear. As such, we aimed at elucidating whether this hyperglycemic effect is due to the dysfunction of pancreatic islet and, if so, the underlying mechanism(s) involved. We investigated the direct effects of niacin on islet function and insulin resistance in HFD-induced obese (DIO) mice and aged mice. Our results showed that eight-week treatments with niacin impaired glycemic control and islet function in DIO and aged mice. Moreover, niacin treatments significantly induced PPARγ and GPR109a expression but decreased SIRT1 expression in pancreatic islets, while islet morphology remained unchanged. In vitro studies showed that niacin decreased glucose-stimulated insulin secretion (GSIS), cAMP, NAD/NADH ratio, and mitochondrial membrane potential (ΔΨm) but increased reactive oxygen species (ROS) transiently, while upregulated expression levels of UCP2, PPARγ and GPR109a in INS-1E cells. In corroboration, the decrease in GSIS and cAMP levels were abolished by the knockdown of GPR109a. These data indicate that chronic treatment of niacin induces hyperglycemia, which is due, partly, to impaired pancreatic islet function, probably via the mediation of islet niacin receptor GPR109a. / Collectively, our study has revealed that inhibition of DPP-4 or SGLT2 alone can improve islet function, and combined inhibition of DPP-4 and SGLT2 works additively to exhibit benefits to islet cell function/morphology, inflammation and insulin resistance, thus improving glycemic control. On the other hand, we have also elucidated that niacin impairs islet β cell function through GPR109a and downstream signaling pathways such as PPARγ and SIRT1. Taken together, the present study has shown the regulation of is let β cell function by different factors, which has an added advance to our knowledge about the intricate relationship between islet function and hyperglycemia and T2DM. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Lihua. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 168-195). / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vii / List of Publications --- p.viii / List of Abbreviations / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Endocrine pancreas --- p.2 / Chapter 1.1.1 --- Structure and composition of endocrine pancreas --- p.3 / Chapter 1.1.2 --- Architecture and composition of the islet --- p.3 / Chapter 1.1.3 --- Endocrine cells and their function --- p.5 / Chapter 1.2 --- Disorders of the endocrine pancreas --- p.9 / Chapter 1.3 --- Insulin --- p.10 / Chapter 1.3.1 --- Insulin Structure --- p.10 / Chapter 1.3.2 --- Insulin actions and insulin receptor --- p.11 / Chapter 1.3.3 --- Insulin secretion --- p.12 / Chapter 1.3.3.1 --- Glucose-induced insulin secretion --- p.13 / Chapter 1.3.3.2 --- Phasic insulin secretion --- p.14 / Chapter 1.3.4 --- The regulation of insulin secretion --- p.16 / Chapter 1.3.5 --- Autocrine insulin feedback --- p.20 / Chapter 1.4 --- Diabetes mellitus --- p.21 / Chapter 1.4.1 --- Type 1 diabetes mellitus (T1DM) --- p.22 / Chapter 1.4.2 --- Type 2 diabetes mellitus (T2DM) --- p.23 / Chapter 1.4.3 --- Obesity and T2DM --- p.23 / Chapter 1.4.4 --- Islet dysfunction and T2DM --- p.25 / Chapter 1.5 --- Incretin hormones and DPP-4 inhibition --- p.27 / Chapter 1.5.1 --- Incretin hormones --- p.27 / Chapter 1.5.2 --- Functions of incretin hormones --- p.30 / Chapter 1.5.3 --- Regulation of GLP-1 --- p.34 / Chapter 1.5.4 --- Incretin-based therapy for T2DM --- p.35 / Chapter 1.6 --- Sodium-dependent glucose cotransporter 2 (SGLT2) and its inhibitors --- p.38 / Chapter 1.6.1 --- Sodium-dependent glucose cotransporter 2 (SGLT2) --- p.38 / Chapter 1.6.2 --- Rationale for SGLT2 inhibition --- p.40 / Chapter 1.6.3 --- Consequences of SGLT2 inhibition --- p.41 / Chapter 1.6.4 --- Strategies of SGLT2 inhibition --- p.43 / Chapter 1.6.4.1 --- SGLT2 inhibitors --- p.44 / Chapter 1.6.4.1 --- SGLT2 inhibitors --- p.47 / Chapter 1.7 --- Niacin (nicotinic acid) and its clinical usage --- p.49 / Chapter 1.7.1 --- Niacin general introduction --- p.49 / Chapter 1.7.2 --- General roles of niacin --- p.49 / Chapter 1.7.3 --- Anti-lipolytic effect --- p.50 / Chapter 1.7.4 --- Niacin receptor --- p.51 / Chapter 1.7.5 --- Hyperglycemic effect of niacin --- p.52 / Chapter 1.8 --- General hypothesis --- p.54 / Chapter Chapter 2 --- General Materials and Methods --- p.56 / Chapter 2.1 --- Experimental animal models --- p.57 / Chapter 2.1.1 --- Animal model of type 2 diabetes --- p.57 / Chapter 2.1.2 --- High-fat diet-induced obese mice --- p.58 / Chapter 2.1.3 --- Aged mice --- p.59 / Chapter 2.2 --- INS-1E cell culture and treatment --- p.59 / Chapter 2.2.1 --- Mouse pancreatic islet isolation --- p.59 / Chapter 2.2.2 --- Primary culture of isolated pancreatic islets --- p.60 / Chapter 2.3 --- Pancreatic islet isolation and culture --- p.60 / Chapter 2.4 --- Glucose-stimulated insulin secretion (GSIS) assay --- p.61 / Chapter 2.5 --- Assessment of glucose homeostasis --- p.61 / Chapter 2.6 --- Determination of mRNA expression --- p.62 / Chapter 2.6.1 --- Design of specific primers --- p.63 / Chapter 2.6.2 --- Total RNA extraction and cDNA synthesis --- p.63 / Chapter 2.6.3 --- Real-time PCR analysis --- p.64 / Chapter 2.7 --- Detection of protein expression --- p.64 / Chapter 2.7.1 --- Western blotting analysis --- p.64 / Chapter 2.7.2 --- Immunofluorescent staining --- p.65 / Chapter 2.8 --- Biochemical analyses --- p.65 / Chapter 2.8.1 --- Plasma insulin and blood HbA1c levels --- p.65 / Chapter 2.8.2 --- Detection of cAMP --- p.66 / Chapter 2.8.3 --- NAD and NADH determination --- p.66 / Chapter 2.9 --- Detection of intracellular ROS --- p.67 / Chapter 2.10 --- Detection of mitochondrial membrane potential --- p.67 / Chapter 2.11 --- Statistical analysis --- p.67 / Chapter Chapter 3 --- Effects of Combining Linagliptin Treatment with BI-38335, A Novel SGLT2 Inhibitor, on Pancreatic Islet Function and Inflammation in db/db Mice --- p.70 / Chapter 3.1 --- Abstract --- p.71 / Chapter 3.2 --- Introduction --- p.72 / Chapter 3.3 --- Materials and Methods --- p.74 / Chapter 3.3.1 --- Animal model and experimental design --- p.74 / Chapter 3.3.2 --- In vivo glucose homeostasis --- p.75 / Chapter 3.3.3 --- Pancreas and islet studies --- p.76 / Chapter 3.3.4 --- Biochemical analyses --- p.77 / Chapter 3.3.5 --- Real-time PCR analyses --- p.77 / Chapter 3.3.6 --- Statistical analysis. --- p.78 / Chapter 3.4 --- Results --- p.78 / Chapter 3.4.1 --- Treatments with DPP-4 and SGLT2 inhibitors lower plasma glucose --- p.78 / Chapter 3.4.2 --- Treatments with DPP-4 and SGLT2 inhibitors improve glycemic --- p.80 / Chapter 3.4.3 --- Pancreatic islet function in db/db mice --- p.83 / Chapter 3.4.4 --- Pancreatic islet and peripheral tissue inflammation --- p.86 / Chapter 3.4.5 --- Islet morphology and preserved beta cells --- p.89 / Chapter 3.5 --- Discussion --- p.93 / Chapter Chapter 4 --- Niacin-Induced Hyperglycemia Is Mediated via Niacin Receptor GPR109a in Pancreatic Islets --- p.98 / Chapter 4.1 --- Abstract --- p.99 / Chapter 4.2 --- Introduction --- p.100 / Chapter 4.3 --- Research design and methods --- p.102 / Chapter 4.3.1 --- Animal model and experimental design --- p.102 / Chapter 4.3.2 --- In vivo glucose homeostasis --- p.102 / Chapter 4.3.3 --- Pancreas and islet studies --- p.103 / Chapter 4.3.4 --- INS-1E cell culture and treatment --- p.103 / Chapter 4.3.5 --- Construction of small interfering RNA for GPR109a --- p.103 / Chapter 4.3.6 --- Real-time PCR analyses --- p.104 / Chapter 4.3.7 --- Western blotting assay --- p.104 / Chapter 4.3.8 --- Detection of intracellular and mitochondrial ROS --- p.105 / Chapter 4.3.9 --- Detection of mitochondrial membrane potential (ΔΨm) --- p.105 / Chapter 4.3.10 --- Measurement of cAMP levels --- p.105 / Chapter 4.3.11 --- Determination of NAD and NADH levels --- p.106 / Chapter 4.3.12 --- Measurement of cell viability --- p.106 / Chapter 4.3.13 --- Statistical analysis --- p.106 / Chapter 4.4 --- Results --- p.106 / Chapter 4.4.1 --- Glycemic control in HFD-induced obese mice --- p.106 / Chapter 4.4.2 --- Pancreatic islet function in HFD-induced obese mice --- p.110 / Chapter 4.4.3 --- Pancreatic islet morphology and gene expression --- p.112 / Chapter 4.4.4 --- INS-1E function and intracellular levels of cAMP, NAD, and NADH --- p.114 / Chapter 4.4.5 --- Gene expression in INS-1E cells --- p.117 / Chapter 4.4.6 --- Status of ROS and ΔΨm in INS-1E cells --- p.119 / Chapter 4.4.7 --- GPR109a knockdown in INS-1E cells --- p.122 / Chapter 4.5 --- Discussion --- p.129 / Chapter Chapter 5 --- Niacin Impairs Pancreatic Islet Glucose-Stimulated Insulin Secretion in Aged Mice through The Suppression of SIRT1 Signaling --- p.134 / Chapter 5.1 --- Abstract --- p.135 / Chapter 5.2 --- Introduction --- p.136 / Chapter 5.3 --- Research design and methods --- p.139 / Chapter 5.3.1 --- Animal model and experimental design --- p.139 / Chapter 5.3.2 --- In vivo glucose homeostasis --- p.139 / Chapter 5.3.3 --- Pancreas and islet studies --- p.140 / Chapter 5.3.4 --- Real-time PCR analyses --- p.140 / Chapter 5.3.5 --- Western blotting assay --- p.140 / Chapter 5.3.6 --- NAD and NADH determination --- p.141 / Chapter 5.3.7 --- NEFA determination --- p.141 / Chapter 5.3.8 --- Statistical analysis --- p.141 / Chapter 5.4 --- Results --- p.142 / Chapter 5.4.1 --- Glycemic control in middle aged mice --- p.142 / Chapter 5.4.2 --- Pancreatic islet function in HFD-induced obese mice --- p.147 / Chapter 5.4.3 --- NAD, NADH levels in pancreatic islet --- p.149 / Chapter 5.4.4 --- Genes expression in pancreatic islet --- p.151 / Chapter 5.5 --- Discussion --- p.150 / Chapter Chapter 6 --- General discussion --- p.156 / Chapter 6.1 --- Combined inhibition of DPP-4 with SGLT2 on islet function, inflammation and insulin resistance in T2DM --- p.158 / Chapter 6.2 --- Niacin impairs islet function in high-fat diet-induced obese mice and aged mice --- p.161 / Chapter 6.3 --- General conclusion --- p.164 / Chapter 6.4 --- Future directions --- p.166 / Chapter Chapter 7 --- Bibliography --- p.167

Orientador: Antonio Carlos Boschero / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T10:55:45Z (GMT). No. of bitstreams: 1 Stoppiglia_LuizFabrizio_D.pdf: 2405428 bytes, checksum: 6387a533bea2ca65f3826092e2220b39 (MD5) Previous issue date: 2006 / Resumo: No diabetes mellitus, radicais de oxigênio estão associados com perda de sensibilidade à glicose ou destruição das células b. Nesse trabalho, investigamos a tolerância de ilhotas de Langerhans de ratos neonatos ao estresse provocado por H2O2, seus mecanismos antioxidantes de defesa e os fatores que promovem manutenção da sensibilidade à glicose. Cultivadas com 1 mM de H2O2, as ilhotas aumentaram até 6' seu consumo de glicose e resistiram ao estresse induzido por H2O2 quando em meio contendo 20 mM de glicose. A expressão da enzima catalase em resposta à glicose se mostrou necessária a essa resistência, mas não suficiente. Em concentrações baixas de H2O2, quando a catalase tem sua atividade modulada por NADPH, observamos que mesmo proteínas sem atividade catalítica adquiriam capacidade antioxidante e eram regeneradas por NADPH. Mapeando a distribuição de atividade peroxidase na ilhota, observamos sensibilidade ao NADPH nas frações nuclear e citossólica. As ilhotas cultivadas em 20 mM de glicose e as que resistem ao H2O2 possuem em comum uma maior atividade da via das pentoses, que gera NADPH citossólico. Nessas ilhotas, verificamos que a produção citossólica de NAD(P)H limita a secreção de insulina. Tais ilhotas produzem NAD(P)H principalmente da oxidação de substratos endógenos no citossol e nas mitocôndrias, ao invés de localizarem seu uso somente no citossol, como se dá nas ilhotas mais sensíveis ao H2O2. A cultura com 20 mM de glicose produziu ilhotas com alta expressão da lançadeira de NADH glicerol-fosfato, enquanto o H2O2 selecionou ilhotas com alta expressão da lançadeira mal/asp. Como ambas as lançadeiras promovem a comunicação entre citossol e mitocôndrias, concluímos que o sistema de lançadeiras e a geração de NADPH sejam fatores críticos para a manutenção da sensibilidade à glicose nas ilhotas / Abstract: In diabetes mellitus, oxygen radicals are associated with loss of glucose-sensibility and destruction of b-cells. In this work, we investigated the tolerance of neonatal rat islets to stress induced by H2O2, the islets antioxidant defense mechanism and factors maintaining islet glucoseresponsiveness. Islets cultured with 1 mM of H2O2 increased 6 fold the glucose uptake and resisted H2O2-induced stress when cultured in media containing 20 mM of glucose. Glucose-induced catalase expression was shown to be necessary to islet cell-survival, although not sufficient. In low H2O2 concentrations, the activity of catalase is dependent on NADPH and we observed that even proteins with no catalytic activity could be antioxidants regenerated by NADPH. Mapping the peroxidase activity in islets, we observed sensibility to NADPH in nuclear and cytossolic fractions. Islets cultured with 20 mM of glucose and islets that survived after culture with H2O2 both showed increased activity of the pentose phosphate pathway, which generate cytossolic NADPH. Is theses islets, we verified that cytossolic production of NAD(P)H limits insulin secretion. Such islets generate NAD(P)H principally from oxidation of endogenous fuels in cytossol and mitochondria, in contrary of the most H2O2-sensible islets which use endogenous fuels exclusively in cytossol. Culture with 20 mM of glucose produced islets with high expression of the glycerol-phosphate NADH shuttle, where as culture with H2O2 selected islets with high expression of the mal/asp shuttle. Since both shuttles promote interchange between cytossol and mitochondria, we have concluded that the shuttle system together with NAD(P)H generation ability are critical factors in maintaining islet glucoseresponsiveness / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Orientador: Everardo Magalhães Carneiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T21:03:55Z (GMT). No. of bitstreams: 1 Batista_ThiagoMartins_M.pdf: 1168177 bytes, checksum: 6c327ab4c396fab9844364b47f69ad79 (MD5) Previous issue date: 2009 / Resumo: A desnutrição ainda é um problema de saúde pública que afeta principalmente países em desenvolvimento e sua prevalência chega a ser crescente em algumas áreas. Vários estudos obtiveram êxito em correlacionar a má nutrição em estágios iniciais de vida com o desenvolvimento de doenças cardiovasculares e diabetes tipo 2 na vida adulta. No modelo de desnutrição pós desmame verifica-se menor secreção de insulina estimulada por glicose e outros agentes insulinotrópicos bem como menor expressão de várias proteínas envolvidas com a funcionalidade da célula b. Estudos realizados por nosso grupo e outros laboratórios mostram que a suplementação de camundongos com taurina aumenta a secreção de insulina além de regular o influxo de íons Ca2+ para as células b, etapa crucial para o processo secretório. Para avaliar os efeitos da taurina sobre animais desnutridos, utilizamos ratos wistar, machos com 21 dias de vida. Os animais receberam dieta contendo 17% de proteína (normoprotéica) (C) ou 6% de proteína (hipoprotéica) (D). Animais C e D receberam suplementação com taurina a 2,5% na água de beber por 30 dias (CT30 e DT30) ou 90 dias (CT90 e DT90). Em seguida avaliamos parâmetros biométricos e bioquímicos, tolerância à glicose, secreção de insulina estimulada por glicose e pelo agonista colinérgico carbacol, expressão de proteínas envolvidas no controle da secreção de insulina e, finalmente, registramos os movimentos citoplasmáticos de íons Ca2+ após estímulo com glicose e carbacol. Verificamos que a restrição protéica retardou o crescimento dos animais além de reduzir a concentração plasmática de proteínas totais (C = 6,81±0,04; CT30 = 7,15±0,54; CT90 = 6,87±0,19; D = 5,35±0,24; DT30 = 5,37±0,28; DT90 = 5,70±0,09 g/dl; n = 3-5) e albumina (C = 3,20±0,11; CT30 = 3,41±0,02; CT90 = 3,18±0,05; D = 2,74±0,07; DT30 = 2,49±0,09; DT90 = 2,67±0,04 g/dl; n = 5-9) sem efeito da suplementação com taurina. Os animais D se mostraram mais tolerantes à glicose e a suplementação com taurina por 90 dias restaurou parcialmente a tolerância desses animais (C = 30249±2682; CT30 = 37255±6691; CT90 = 29365±2257; D = 16916±1609; DT30 = 18791±2859; DT90 = 23425±3856 AAC; n = 5-9). Nesse trabalho mostramos que a suplementação com taurina corrige a hipoinsulinemia verificada em animais desnutridos alimentados (C = 4,97±0,34; CT90 = 3,56±0,52; D = 1,39±0,10; DT90 = 3,31±0,70 ng/ml; n = 5-8) bem como a responsividade de ilhotas isoladas a concentrações crescentes de glicose. Verificamos também que a taurina normaliza a secreção de insulina potencializada pelo carbacol (C = 9,4+0,8; CT90 = 12,4+0,7; D = 6,4+0,5; DT90 = 9+0,7 ng/ml; n = 12). As respostas secretórias foram observadas em conjunto com a regulação da expressão das proteínas SERCA3 (C = 100+21; CT90 = 174+17; D =96+90; DT90 = 149+11 % do C; n = 6), receptor muscarínico M3 (C = 100+24; CT90 = 155+80; D = 51+10; DT90 = 108+14 % do C; n = 5) e sintaxina 1 (C = 100+30; CT90 = 92+40; D = 50+12; DT90 = 77+11 % do C; n = 5) que participam do controle de diferentes etapas do processo de secreção de insulina. Por fim, verificamos que a suplementação com taurina melhorou o padrão de oscilação de íons Ca2+ após estímulo com glicose. Concluímos então que a suplementação com taurina por 90 dias restaura a sensibilidade das ilhotas à glicose e ao carbacol possivelmente pela regulação do fluxo de cálcio para as células b bem como pela modulação da expressão de proteínas que controlam o processo de secreção de insulina. / Abstract: Malnutrition still is a public health issue, especially in developing countries. Many studies correlate malnourishment during early life and the development of cardiovascular disease and type 2 Diabetes Mellitus on latter stages. Animal models of malnutrition reveal impaired insulin secretion stimulated by glucose and other insulinotropic agents as well as lower expression of key proteins for b cell function. The literature shows that taurine supplementation increases insulin secretion and regulates calcium dynamics on b cells. Male, 21 days old, wistar rats received diet containing 17% (C) or 6% (D) of protein. Both groups received taurine supplementation on the drinking water for 30 (CT 30 and DT 30) and 90 (CT90 and DT 30) days. Next we assessed biometric and biochemical parameters, glucose tolerance, glucose and carbacholstimulated insulin secretion, protein expression of muscarinic M3 receptor, Phospholipase C b2, SERCA3, Syntaxin 1 and, finally, we registered cytoplasmic Ca2+ after stimulus with glucose and carbachol. Protein restricted rats showed lower body weight, plasma proteins (C = 6,81±0,04; CT30 = 7,15±0,54; CT90 = 6,87±0,19; D = 5,35±0,24; DT30 = 5,37±0,28; DT90 = 5,70±0,09 g/dl; n = 3-5), albumin (C = 3,20±0,11; CT30 = 3,41±0,02; CT90 = 3,18±0,05; D = 2,74±0,07; DT30 = 2,49±0,09; DT90 = 2,67±0,04 g/dl; n = 5-9) and increased glucose tolerance (C = 30249±2682; CT30 = 37255±6691; CT90 = 29365±2257; D = 16916±1609; DT30 = 18791±2859; DT90 = 23425±3856 AUC; n = 5-9). Taurine supplementation had no effect upon nutritional status parameters and partially restored glucose tolerance and insulinemia to C levels. Taurine increased secretory response to glucose and carbachol (C = 9,4+0,8; CT90 = 12,4+0,7; D = 6,4+0,5; DT90 = 9+0,7 ng/ml; n = 12). It also increased protein expression of M3 receptor (C = 100+24; CT90 = 155+80; D = 51+10; DT90 = 108+14 % of C; n = 5), SERCA 3 (C = 100+21; CT90 = 174+17; D =96+90; DT90 = 149+11 % of C; n = 6) and syntaxin 1 (C = 100+30; CT90 = 92+40; D = 50+12; DT90 = 77+11 % of C; n = 5). Finally, taurine supplementation for 90 days improved Ca2+ dynamics when the islets were stimulated with glucose. In conclusion, these data show that taurine supplementation restores secretory responsiveness to glucose and carbachol possibly through Ca2+ dynamics modulation and increased expression of key proteins for insulin secretion. / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Orientador: Antonio Carlos Boschero / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T00:11:07Z (GMT). No. of bitstreams: 1 Vanzela_EmerielleCristine_D.pdf: 2535043 bytes, checksum: eee7b54cea5f470c5637de8c4b3385f5 (MD5) Previous issue date: 2010 / Resumo: A obesidade atingiu proporções alarmantes constituindo-se num fator de risco para o desenvolvimento de várias doenças. O aumento da resistência periférica à insulina acompanha esta patologia e a incapacidade da célula beta pancreática em suprir a maior necessidade por insulina leva ao desenvolvimento de intolerância à glicose, hiperglicemia e diabetes. Por esta razão, é importante investigar mecanismos que tornem a célula beta capaz de aumentar sua capacidade secretória. A exemplo da obesidade, resistência periférica à insulina é também observada durante a prenhez. No entanto, neste caso, a célula beta é capaz de aumentar a produção e secreção do hormônio, mantendo a tolerância à glicose em condições adequadas. Diante disso,decidimos investigar a sensibilidade à insulina e a consequente resposta das células beta pancreáticas durante a prenhez em ratas obesas. Observamos que a alimentação com a dieta de cafeteria aumentou o ganho de peso, bem como os depósitos de gordura das ratas. Ratas obesas não-prenhes (Caf) e prenhes (CafP) apresentaram tolerância à glicose diminuída, associada a um aumento da insulina plasmática em resposta à sobrecarga de glicose no grupo CafP. Apesar disso, as glicemias de jejum e pós-prandial foram normais nos dois grupos. No entanto, as ratas Caf e CafP apresentaram hiperinsulinemia (jejum e alimentado), aumento do índice insulina/glicose e do AGL plasmático (alimentado). Ainda, houve redução na sinalização da insulina no fígado e músculo esquelético das ratas Caf e CafP, aos 15 e aos 19 dias de prenhez, de forma mais exacerbada do que a redução observada nas ratas controle prenhes. Em paralelo, as ilhotas isoladas das ratas Caf secretaram menos insulina em resposta a diferentes estímulos. Contudo, o conteúdo total de insulina, a secreção estimulada por PMA (ativador da PKC), a produção decompostos redutores e a oxidação de glicose, na presença de 11,1 mmol/L do açúcar, foram similares entre as ratas Caf e as controle não-prenhes. Entretanto, as ilhotas isoladas das ratas Caf apresentaram redução na mobilização do Ca2+ citoplasmático livre frente à glicose ou tolbutamida, acompanhada pela redução da expressão gênica da subunidade ?1.2 do canal de cálcio voltagem-dependente (CaVa1.2), e da Ca2+- ATPase do retículo endoplasmático tipo 2a. Independente da dieta, a prenhez aumentou a secreção de insulina em resposta à glicose, a produção de compostos redutores, a oxidação de glicose, a amplitude e a frequência das oscilações do Ca2+ citoplasmático e, a expressão gênica do CaVa1.2. Concluindo, a prenhez nas ratas obesas melhorou o manejo do Ca2+ e restaurou a secreção de insulina por ilhotas isoladas. Contudo, esta restauração não foi suficiente para vencer o aumento da resistência periférica à insulina e normalizar a tolerância à glicose nas ratas obesas / Abstract: The incidence of obesity reached alarming levels worldwide. This illness constitutes a risk factor for the development of several other diseases. The augmented peripheral insulin resistance accompanies this pathology, and the failure of the pancreatic beta cell to overcome the higher demand for insulin causes glucose intolerance, hyperglycemia and diabetes. For this reason, it became interesting to investigate mechanisms that make the beta cell capable to increases its secretory capacity. As obesity, peripheral insulin resistance is also observed during pregnancy. Nevertheless, in this situation, the beta cell is capable to enhance insulin production and release, maintaining glucose tolerance at adequate levels. Therefore, we decided to investigate insulin sensibility and the consequent beta cell response during pregnancy in obese rats. We observed that cafeteria diet enhanced weight gain and fat pads in rats. Despite no differences were noticed in obese non-pregnant (Caf) and pregnant (CafP) rats, during fast and fed states, the glucose tolerance was diminished in these rats, associated with an augmented plasma insulin levels in response to a glucose load inCafP rats. However, Caf and CafP rats had hyperinsulemia (fast and fed), higher insulin/glucose index, and enhanced plasma FFA (fed state). In addition, we observed a reduction in insulin signaling in liver and skeletal muscle from Caf and CafP rats, at15th and 19th days of pregnancy, higher than that registered in control pregnant rats. Also, there was a reduction in insulin secretion induced by different stimuli in is lets from Caf rats. However, total islet insulin content, PMA-stimulated insulin secretion, production of reducing equivalents, and glucose oxidation in the presence of 11.1mmol/L glucose, were similar between islets from Caf and non-pregnant control rats .Nevertheless, glucose- and tolbutamide-induced Ca2+ mobilization, a1.2 subunit of thevoltage sensitive Ca2+ channel (CaVa1.2), and sarcoendoplasmic reticulum Ca2+ATPase 2a gene expression were reduced in islets from Caf rats. Independently of the diet, pregnancy enhanced glucose stimulated insulin secretion, reducing equivalents production, glucose oxidation, amplitude and frequency of cytoplasm Ca2+ oscillations, and CaVa1.2 gene expression. In conclusion, although pregnancy improved Ca2+ handling and restored insulin secretion in cafeteria diet-induced obese rats, this restoration was not enough to overcome the increase in peripheral resistance and normalize glucose tolerance in these obese rats / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Poon, Chui Wa Christina. / "August 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 123-131). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 論文摘要 --- p.vi / ACKNOWLEDGEMENTS --- p.ix / PUBLICATIONS --- p.x / Abstracts --- p.x / ABBREVIATIONS --- p.xii / Chapter 1. --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1. --- Diabetes --- p.1 / Chapter 1.1.1. --- Overview --- p.1 / Chapter 1.1.2. --- Diagnostic Criteria of Type-2 Diabetes --- p.2 / Chapter 1.1.3. --- Type-2 Diabetes (T2DM) --- p.3 / Chapter 1.1.3.1. --- Impaired Insulin Synthesis and Insulin Secretory Defects in Type-2 Diabetes --- p.3 / Chapter 1.1.3.2. --- β-Cell Dysfunction --- p.5 / Chapter 1.1.3.3. --- Insulin Resistance --- p.5 / Chapter 1.1.4. --- Glucose Toxicity --- p.6 / Chapter 1.1.4.1. --- Fasting Hyperglycemia --- p.8 / Chapter 1.1.4.2. --- Postprandial Hyperglycemia --- p.8 / Chapter 1.2. --- Oxidative Stress --- p.8 / Chapter 1.2.1. --- ROS and Mitochondria --- p.8 / Chapter 1.2.2. --- ROS Production by Mitochondria --- p.9 / Chapter 1.2.3. --- The Relationship of Glucose Recognition by β-cells and Oxidative Stress --- p.11 / Chapter 1.2.4. --- Important Roles of Glutathione in Pancreatic β-cells and Glutathione Synthesis --- p.14 / Chapter 1.2.5. --- N-acetyl-L-cysteine - A Potential Drug Treatment for Type-2 Diabetes? --- p.17 / Chapter 1.3. --- Role of F-actin Cytoskeleton on Glucose-induced Insulin Secretion --- p.18 / Chapter 1.4. --- Current Clinical Treatments for Type-2 Diabetes Mellitus --- p.21 / Chapter 1.4.1. --- Metformin --- p.22 / Chapter 1.4.2. --- Sulfonylureas --- p.22 / Chapter 1.4.3. --- Thiazolidinediones --- p.23 / Chapter 1.4.4. --- Glinides (Meglitinide Analogues) --- p.23 / Chapter 1.4.5. --- α-Glucosidase (AG) Inhibitors --- p.24 / Chapter 1.4.6. --- Dipeptidyl Peptidase-4 (DPP-4) Inhibitors --- p.24 / Chapter 1.4.7. --- (Clinical) Antioxidant Treatment --- p.24 / Chapter 1.5. --- Animal Models Used in Type-2 Diabetes Research --- p.25 / Chapter 1.6. --- Aims of Study --- p.27 / Chapter 2. --- RESEARCH DESIGN & METHODS --- p.28 / Chapter 2.1. --- Materials --- p.28 / Table 1. Sources and concentrations of drugs tested in this study: --- p.28 / Culture Medium - --- p.29 / General Reagents --- p.29 / Chapter 2.2. --- Isolation of Islets of Langerhans and Single Pancreatic β-Cells --- p.31 / Chapter 2.3. --- Measurement of Mitochondrial ROS Levels --- p.32 / Chapter 2.4. --- Measurement of Islets Insulin Release and Insulin Content --- p.34 / Chapter 2.4.1. --- Preparation of Samples --- p.34 / Chapter 2.4.2. --- Enzyme-Link Immunosorbent Assay (ELISA) --- p.35 / Chapter 2.5. --- Immunocytochemistry --- p.35 / Chapter 2.6. --- Data and Statistical Analysis --- p.37 / Chapter 3. --- RESULTS --- p.38 / Chapter 3.1. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on Releasable Insulin Levels and Insulin Contents in Response to Low Glucose (5 mM) and High Glucose (15 mM) of Isolated Pancreatic Islets of (db+/m+) and (db+/db+) Mice" --- p.38 / Chapter 3.1.1. --- Effect of L-NAC on Insulin Secretion and Insulin Contents --- p.38 / Chapter 3.1.2. --- Effect of Cytochalasin B on Insulin Secretion and Insulin Contents --- p.39 / Chapter 3.1.3. --- Effect of 4-Phenyl Butyric Acid on Insulin Secretion and Insulin Contents --- p.43 / Chapter 3.1.4. --- Effect of Ursodeoxycholic Acid on Insulin Secretion and Insulin Contents --- p.46 / Chapter 3.1.5. --- Effect of Hydrogen Peroxide on Insulin Secretion and Insulin Contents --- p.49 / Chapter 3.1.6. --- Effect of Jasplakinolide on Insulin Secretion and Insulin Contents --- p.53 / Chapter 3.1.7. --- Effect of Thapsigargin on Insulin Secretion and Insulin Contents --- p.57 / Chapter 3.1.8. --- Effect of BSO on Insulin Secretion and Insulin Contents --- p.61 / Chapter 3.2. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on Mitochondrial ROS Levels in Response to High Glucose (15 mM) Challenge in Isolated Single Pancreatic β-Cells of (db +/m+) and (db +/db +) Mice" --- p.65 / Chapter 3.2.1. --- "Effects of L-NAC (20 mM), 4-Phenyl Butyric Acid (4-PBA) (1 mM), Ursodeoxycholic Acid (UA) (500 μg/ml), H202 (200 μM), Thapsigargin (0.5 μM) and DL-Buthionine-[S,R]-Sulfoximine (BSO) (0.1 μM) Pre-treatments on Mitochondrial ROS Level in Response to High Glucose (15 mM) Challenge" --- p.65 / Chapter 3.2.2. --- "Effects of L-NAC (20 mM), Cytochalasin B (10 μM) and Jasplakinolide (5 μM) Pre-treatments on Mitochondrial ROS Level in Response to High Glucose (15 mM) Challenge_" --- p.76 / Chapter 3.3. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on F-actin Cytoskeleton Levels Incubated in Low Glucose (5 mM) and High Glucose (15 mM) Medium in Single Pancreatic β-Cells of Non-Diabetic (db +/m+) and Diabetic (db +/db +) Mice" --- p.81 / Chapter 4. --- DISCUSSION --- p.100 / Chapter 4.1. --- General Discussion --- p.100 / Chapter 5. --- SUMMARY --- p.120 / Chapter 6. --- FUTURE PERSPECTIVES --- p.121 / Chapter 7. --- REFERENCES --- p.123